Extraction of nettle (Urtica dioica L.) toxins under natural biting conditions

A group of natural poisons from various animals, plants and microorganic sources can be extracted, produced and processed. Following ten years of field and laboratory research and studies, resulted from the creation of the first live collection of Iranian nettle ecotypes (LCINs) at the University of Zanjan, the feasibility of fresh and live extraction of nettle poison in pristine and untouched conditions was examined. In this study, the ability of tree tissues to absorb, hunt and sink nettle hairs, including styrofoam, nanofabric and sponge of the same length (15 cm) and same diameter (4 cm) having the same size of pores, was studied in four selected nettle ecotypes, including ecotypes of Mashhad, Mazandaran, Gilan and Zanjan provinces, Iran. For all four ecotypes on the three studied surfaces, the mean number of fully stuck and sunken needles, broken and sunken needles on the surface tissue, pores torn by plant needles and pores containing pale green liquid were counted and fully scrutinized. The results showed that sponges can be a suitable texture for hunting nettle hairs for extracting fresh and raw live venom of approximately 5 ml on a sponge source for 5 min. Based on GCMS analysis of total venom extraction resulting profile from the studied protocols had more than 10 compounds including some important sulfur containing such as: 2,2-dimethyl-propyl 2,2-dimethylpropanesulfinyl sulfone and 2-ethylthiolane, S,S-dioxide, etc. In this method, there is no need to remove the plant and stem. Its unique advantage is in continuous poison harvests during the 6-month growing season. Based on published research, this is the first report of live extraction of nettle medicinal poison.


Scientific Reports
| (2022) 12:5917 | https://doi.org/10.1038/s41598-022-09916-0 www.nature.com/scientificreports/ NaOH 13 . Drying the plant limbs or cooking it with heat not only removes the biting property but it also destroys or changes the nature of the material in the nettle bite 14 . Hence, introducing a method for natural nettle venom collection from live plants (without plant harvesting) is necessary for industrial extraction of medicinal matter from this valuable plant. The main incentive of this research is to establish an efficient and practical method for collecting of nettles venom on natural bite conditions. Based on published research reports, this is the first study to scientifically focus on raw and natural nettle venom.

Materials and methods
Experimental site. The experimental site was located at latitude 36° 68′ N, longitude 48° 38′ E, and an altitude of 1579 m with a cold climate, a mean annual precipitation of 295 mm, and a mean annual temperature of 10 °C. The average minimum temperature in the coldest month is "Bahman to -7.5", and the average maximum temperature in the warmest month is "August, 32.1". During the year, the temperature drops below zero for 118 days, with January and February ranking first with 27 days. The wettest month is May with 52.5 mm, and the driest month is September with 3.5 mm. The maximum recorded wind speed of 27 m per second was "97 km per hour". The prevailing wind in Zanjan in most months of the year was in the east, and the average wind speed of 3 m per second was "11 km per hour". Plant material. The academic permission for collecting and researching medicinal plants was obtained from Head of Biotechnology, Department Research Institute of Modern Biological Techniques, University of Zanjan, Zanjan, Iran. The study complies with all relevant guidelines. All experiments were performed on 20 nettle ecotypes in the National Live Collection of Iranian Nettle (LCINs), located at the Research Institute of Modern Biological Techniques, University of Zanjan, Zanjan, Iran. A total of 4 nettle ecotypes (4 different provinces of Iran), similar in age and growth morphological conditions, were randomly selected and further used to collect nettle bites and venom. These nettle ecotypes were collected from different nettle habitats in Iran from different provinces in Iran, including Mazandaran, Gilan, Mashhad and Zanjan. These collections were cultivated in both greenhouse and field conditions and were raised and maintained (2013-2022) (Fig. 1).
Morphology study of explants. All stages of hair penetration in to 3 different surfaces were analyzed during the research process, and sample stems, leaves and hairs were documented with a stereoscopic microscope (Nikon SMZ1, Tokyo, Japan). Photographs were taken with an Olympus microscope (Olympus CX31, Tokyo, Japan) at 40 and 400 magnifications.

Collection of poison.
Three small porous circular plates with a length of 15 cm and a diameter of 4 cm in three types of styrofoam, nanofabric and sponge of the same length (15 cm) and same diameter (4 cm) were embedded on a light rod and so that they could be rotated on the rod in accordance with the rod axis.
GCMS analysis. GC-MS analysis of the extract was performed via an Agilent 7890B (USA) gas chromatograph equipped with an HP5-MS (5% phenyl/95% dimethylpolysiloxane) capillary column (60 m × 0.25 mm 1D × 0.25 μm). This was coupled with a 5977A mass spectrometer in the electron impact (EI) ionization mode with an ionizing energy of 70 eV. . Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1 ml/min, and an injection volume of 1 μl was employed (split ratio of 1:20 ) under following conditions:injector temperature 280 °C, ion-source temperature 230 °C and MS transfer line of 300 • C. The oven temperature was programmed from 70 °C (isothermal for 5 min.), with an increase from 15 °C/min to 200 °C and then 8 °C/min going up to 280 °C, and held for 10 min. A full scan interval of 0.5 s and fragments from 45 to 600 amu (Da) was

Results
The photos below show the live ecotypes of nettles in the Live Collection of Iranian Nettles (LCINs) located at the University of Zanjan with more than 50 nettle ecotypes. These plant specimens were studied under both greenhouse and field living conditions. This collection has been established since 2013 (Figs. 2, 3). Using a oneand-a-half meter innovative rod in which the three studied surfaces, including Styrofoam, Nanofabric and Sponge as circular and rolling surfaces, were randomly installed and designed in the same size and dimensions for two  www.nature.com/scientificreports/ minutes, which were completely and slowly performed on the leaves and stems (Fig. 1). Each of the rotating cylindrical surfaces had a side area of 376.8 cm 2 and a final volume of 11.7 cm 3 , but the volume of the sponge mass had to be reduced from the maximum total amount. Ecotypes have just been contacted. Immediately after contact, the surfaces used were immediately placed under a stereomicroscope, which was located in the same place, and consequently the desired surfaces were carefully evaluated and photographed. For all four ecotypes on the three surfaces studied, the mean number of fully stuck and sunken needles, broken and sunken needles on the surface tissue, pores torn by plant needles and pores containing pale green liquid were counted and noted (Figs. 4,5,6,7,8,9). The results of surveys and data analysis are given in Table 1. Based on Fig. 10. Total venom extraction resulting profile from the studied protocols had more than 10 compounds including some important sulfur containing such as: 2,2-dimethyl-propyl 2,2-dimethylpropanesulfinyl sulfone and 2-ethylthiolane, S,S-dioxide, etc. The main components were presented in Table 2.
The results showed that sponges can be a suitable texture for hunting nettle hairs for extracting fresh and raw venom. The production of graphs in three replications, as described in Fig. 10, shows the scientific and executive success of the presented protocol. Based on published research, this is the first report of live extraction of nettle medicinal poison.

Conclusion
Based on the statistical analysis of the data, the cylindrical plate of the sponge has more communication and connection power and hunting of fine nettle hairs than other levels, and it is suggested and introduced for additional experiments on the extraction and purification of nettle venom. Another advantage of this porous texture (sponge) is that it easily penetrates the water and easily loses the absorbed water. This convenience and  www.nature.com/scientificreports/ ease of water loss in nanofabric is much less, and basically not much water is absorbed in the foam. This valuable physical property of the sponge is better for extracting the active ingredients that are involved and accumulate in the pores of the tissue with a suitable solvent such as water.